Treating patients infected with Human Immunodeficiency Virus-1 (HIV-1) is a substantial clinical challenge with evolving pharmaceutical drug and dosing options. Treatments should improve the health and quality of life for HIV-infected individuals by reducing viremia and preserving immunological function. Currents therapeutic approaches utilize combination antiretroviral drug regiments (cARV, historically referred to highly active antiretroviral therapy, or HAART) to reduce ongoing viral replication and improve the patient's prognosis. However, cARV therapies are not curative and furthermore they are expensive, are associated with high rates of drug-related toxicity, and are highly susceptible to viral evasion through resistance-conferring mutation. Genetic targeting CCR5 is a promising alternative approach to cARV therapy. The CCR5 gene is used by HIV-1 for cellular entry and those individuals who naturally lack CCR5 expression are fully resistant to HIV-1 infection. In an important clinical case study, bone marrow transplantation from a CCR5-deficient donor has produced the first recorded case of functional cure of an ongoing HIV-1 infection. This has prompted therapeutic strategies to create HIV-1 resistant patient-derived T cells using nuclease- based reagents to create disruptive mutations in the CCR5 gene. Though providing critical proof-of- concept in HIV-1 patients, the success of the gene-targeting therapy currently in clinical trials appears to be limited due to low gene disruption activity of these firt-generation nuclease technologies. Pregenen has used its surface display engineering platform to produce highly specific CCR5- targeting LAGLIDADG homing endonucleases (LHE), recently reformatted as extremely powerful MegaTAL nucleases and combined with an additional layer of rate-enhancement technology - exonuclease-accelerated genome editing (X-AGE) developed at SCRI. The proposed therapy will isolated and process patient-derived T-cells by delivering these enhanced reagents using a simplified and scalable mRNA-based method. This manufacturing process will be used to create HIV-resistant, CCR5-deficient autologous CD4+ T cell. Autologous CCR5-disrupted T cells have the potential to durably suppress HIV-1 infection and reconstitute the full functionality of the CD4+ compartment while reducing dependence on antiretroviral drugs. The experiments described in this proposal are part of a comprehensive plan to bring the CCR5-targeting nuclease therapy to the clinic. We plan to profile the safety, biodistribution, and potency of primary human T cells treated with CCR5-targeting reagents. The best-performing reagents and protocols will be used to generate CCR5-deficient human CD4+ T cells to test the capacity of these cells to withstand HIV infection in vitro and in vivo. We will se this data-set to support pivotal preclinical experiments and subsequent IND submission for the continued clinical development of our cellular therapy.